Journal o/ Neurocherni~tryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Functional Role of Amino-Terminal i‘6 and Serine27of Ga~in Receptor and Effector Coupling

Maurice K. C. Ho and *yung H. Wong

Department of Biology and *Biotechnology Research Institute, Hong Kong University of Science and Technology,

Clear Water Bay, Kowloon, Hong Kong

Abstract: The a subunit of G. (a‘) harbors two N-terminalserine residues (at positions 16 and 27) that serve asprotein kinase C-mediated phosphorylation sites. Thecognate residues in the a subunit of G~

1provide bindingsurfaces for the ßi subunit. We used three serine-to-ala-nine mutants of a~to investigate the functional impor-tance of the two N-terminal serine residues. Wild-typeor mutant a~was transiently coexpressed with differentreceptors and adenylyl cyclase isozymes in human em-bryonic kidney 293 cells, and agonist-dependent regula-tion of cyclic AMP accumulation was examined in a set-ting where all endogenous a subunits of G were inacti-vated by pertussis toxin. Replacement of one or bothserine residues by alanine did not alter the ability of a,to interact with ô-opioid, dopamine D2, or adenosine A1receptors. Its capacity to inhibit endogenous and type VIadenylyl cyclases was also unaffected. Functional releaseof fly subunits from the mutant a, subunits was not im-paired because they transduced fly-mediated stimulationof type Il adenylyl cyclase. Constitutively active mutantsof all four a, subunits were constructed by the introduc-tion of a Q205L mutation. The activated mutants showeddifferential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormonereceptor-transfected cells. Loss of both serine residues,but not either one alone, impaired the receptor-indepen-dent inhibition of adenylyl cyclase by the GTPase-defi-cient mutant. Thus, replacement of the amino-terminalserine residues of a~has no apparent effect on receptor-mediated responses, but these serine residues may beessential for ensuring transition of a, into the active con-formation. Key Words: Adenylyl cyclase—G proteins—Phosphorylation—Signal transduction.J. Neurochem. 68, 2514—2522 (1997).

(Ammer and Schulz, 1994) with peripheral distribu-tions in platelets (Canson et al., 1989) and pancreaticislet cells (Zigman et al., 1994). Many G-coupledreceptors canfunctionally interact with G, and performPTX-insensitive signaling events (Wong et al., 1992;Lai et al., 1995; Shum et al., 1995; Tsu et al., 1995a,b;Yung et al., 1995). Like other a subunits,a, undergoesextensive covalent modifications at its N-terminus.These include N-myristoylation (Mumby et al., 1990),palmitoylation (Linder et al., 1993), arachidonylation(Hallak et al., 1994), and protein kinase C (PKC)-mediated phosphorylation (Lounsbury et al., 1991).

It has become increasingly apparent that covalentmodifications on the N-termini of a subunits may bearimportant functional significance. For example, fattyacylation-deficient a, mutants generated by site-di-rected mutagenesis exhibit altered inhibitory actionson AC activity (Wilson and Boume, 1995). More in-teresting is that PKC-mediated phosphorylation of a,prevents its association with fly subunits (Fields andCasey, 1995). Of the two known PKC-targeted serineresidues (Lounsbury et al., 1993), Ser‘

6 is conservedwithin the G

1 subfamily. The cognate serine residue ispredicted to form a hydrogen bond with Lys

89 of thefi subunit in the crystal structure of a heterotrimermade up of a G

1 a subunit (a11 )Ia~1chimera and ß1y1

subunits (Lambright et al., 1996). The other PKC-targeted serine residue (5er

27) is unique for a,. Thecorresponding residue (Ala23) in the a

11/a~1chimeraappears to interactwith Leu

55 of the ß~subunit through

G, [orG.(Fong et al., 1988; Matsuokaetal., 1988)]is a heterotrimeric (afly) guanine nucleotide-bindingregulatory protein (G protein) whose a subunit (a,)can inhibit adenylyl cyclases (ACs) to the same extentas those produced by the better known subtypes of thea subunit of G~(ai) (Wong et al., 1992; Kozasa andGilman, 1995). Unlike a

1, a, is resistant to pertussistoxin (PTX)-catalyzed ADP-ribosylation and is pri-marily expressed in neuronal tissues and cell lines

Received October 7, 1996; revised manuscript received January7, 1997; accepted January 22, 1997.

Address correspondence and reprint requests to Dr. Y. H. Wongat Department of Biology, Hong Kong University of Science andTechnology, Clear Water Bay, Kowloon, Hong Kong.

Abbreviations used: A1R, adenosine A1 receptor; AC, adenylylcycla.se; a, a,, a5, a,, a~,and a,, a subunit of G,, G,, G5, G,, G.and G,, respectively; aWT, wild-type a subunit; cAMP, cyclicAMP; D,R, dopamine D, receptor; ÔOR, b-opioid receptor; DPDPE,[D-Pen

2,~-Pen5]enkephalin; GTPyS, guanosine 5 ‘-0- ( 3-thiotri-phosphate); hCG, human choriogonadotropin; HEK293 ce!Is. humanembryonic kidney 293 cells; IBMX, l-methyl-3-isobutylxanthine;LHR, luteinizing hormone receptor; MEM, minimal essential me-dium; PKC, protein kinase C; PTX, pertussis toxin.

2514

ROLE OF SER‘6 AND SER27 IN Ga,. FUNCTIONS 2515

van der Waals forces. Both serine residues on the N-lerminal helix of a, are thus predicted to lie on the flycontacting surface and may be crucial for the efficienthinding and release of fly subunits. Furthermore, theN-terminus may stabilize the C-terminal a-helix andaffect the nucleotide binding or other characteristics ofdifferent a subunits (Denker et al., 1995). The crystalstrLlcture of a

11 showed that both termini of the a poly-pcptide are in close proximity to each other and mayeven lorm an independent structural domain (Wall etal., 1995). These findings support the postulation thatlhe N-terminus of a G protein a subunit may be in-volved in receptor coupling, subunit interaction, andellector coupling.

Because PKC-mediated phosphorylation of a, im-pairs its association with fly subunits (Fields andCasey. 1995) and both Ser© and 5er

27 are putativehinding sites for fly, it seems plausible that the twoserine residues may serve as regulatory switches forG,-mediated signaling. To explore the functional sig-nilicance of the two PKC-targeted serine residues ofa,., we used a, mutants where 5er16, Ser27, or bothresidues were replaced by alanine. Substitution of thetwo N-terminal serine residues by alanine is expectedto abate a, signaling, especially if the hydroxyl groupson the two serines are required for the binding of fly.‘I‘he a, mutants were tested for their capacity to interactwith receptors as well as to regulate different AC iso-/ymes in a transient expression system. Direct regula-tion of ACs by the a, mutants was further examinedhy introduction of a GTPase-deficient mutation at co-don 205 (replacement of GIn by Leu). It is interestingthat the triple mutant a,16/27QL was unable to inhibit

constitutively cyclic AMP (cAMP) accumulation. Ourresults suggest that although the N-terminal serine resi-dues of a, have no apparent effect on receptor coupling

and subunit dissociation of G,, they may somehowassist in the facilitation or maintenance of the activeconlormation of a,.

MATERIALS AND METHODS

MaterialsThe human wild-type a, (a,WT) and phosphorylation-

resistant iliutant a, cDNAs, a,lb, a,27, and a,l6/27 (Louns-htiiy et al., 1993). were generously provided by David R.Manning (University of Pennsylvania School of Medicine,Philadelphia, PA, U.S.A.). The eDNA encoding type VI ACwas a kind donation by Ravi lyengar (Mount Sinai Schoolt,l Medicine, New York, NY, U.S.A.). The a,-specific anti-serum l‘-961 (Casey et al., 1990) was a gift from Patrick(‘asey (Duke University Medical Center, Durham, NC,LISA.). PTX was purchased from List Biological Labora-tories (Camphell. CA, U.S.A.). Human embryonic kidney293 (11EK293) cells were obtained from the American Type(‘ullurc Collection (ATCC CRL- 1573). [3H1 Adenine was

purchased froni Amersham Corp. Plasmid purification col-tnnns were obtained from Qiagen. Cell culture reagents weretthtaincd from Life Technologies (Grand Island, NY,LISA.). and all other chemicals were purchased from

g lia.

Construction of GTPase-deficientphosphorylation-resistant ~.

GTPase-deficient mutants of wild-type and phosphoryla-tion-resistant a, subunits were constructed by exchangingparts of the sequences of these human cDNAs with rat a,QLeDNA. The human and rat homologues have 98.3% aminoacid identity and are functionally indistinguishable. A 1.0-kb Xcml/XhaI fragment of the rat a,QL-pcDNAl (Wong etal., 1992) containing the Q205L point mutation was isolatedand used to replace a corresponding fragment of a,WT inpDP5 (Lounsbury et al., 1993). Subsequent constructionsof GTPase-defieient phosphorylation-resistant a, eDNAs(denoted as a, I 6QL, a,27QL, and a, 16/ 27QL) were accom-plished by substituting a 1.0-kb Smal fragment from a, 16,a,27, or a,16/27 with the corresponding region in a,QL-pDP5. Resultant constructs were verified by multiple restric-tion digests.

Transfection of HEK293 cellsHEK293 cells were cultured with Eagle‘s minimal essen-

tial medium (MEM) supplemented with 10% (vol/vol) fetalcalf serum, 50 units/mI penicillin, and 50 pg/mI streptomy-cin at 37°Cin humidified air with 5% CO

2. For cAMP assay,cells were seeded onto 12-well plates at 2 Y l0~cells perwell the day before transfection. DEAE-dextran-mediatedtransfection was performed as described previously (Wong,1994). In brief, appropriate amounts of various DNA sam-pies purified by Qiagen column chromatography were mixedwith growth medium containing 400 pg/nil DEAE-dextranand 100 pM chloroquine. Cells were incubated with thetransfection cocktails for 1.5—2.0 h and then shocked for Imin at room temperature in phosphate-buffered saline con-taining 10% (vol/vol) dimethyl sulfoxide. After rinsing withphosphate-buffered saline, the cells were returned to growthmedia for 24 h. Approximately 50% of the cell populationwill take up the eDNAs as indicated by cotransfecting aplasmid DNA encoding fi-galactosidase as a reporter.

cAMP accumulationTransfected HEK293 cells were labeled with [

3Hjadenine(I pCi/ml) in MEM containing 1% (vol/vol) fetal calf se-rum I day after transfection. PTX (100 ng/ml) was addedwhere appropriate. After 18—24 h the labeling media werereplaced by I ml of assay medium (20mM HEPES-bufferedMEM) containing 1 mM 3-isobutyl-l-methylxanthine (orRo-20-1724 for experiments using adenosine A

1 receptor)and the appropriate drugs. The cells were incubated at 37°Cfor 30 min and then lysed with I ml of ice-cold 5% (wt/vol) trichloroacetie acid containing 1 mM ATP at 4°C for30 min. [

3H IcAMP was extracted t‘rom the pool of labelednucleotides by sequential ion-exchange chromatography asdescribed previously (Wong, 1994). The cAMP levels wereinterpreted as the ratios of the cpm of [3H IcAMP fractionsto those 01‘ the total labeled nucleotide fractions and ex-pressed as [cAMP/(cAMP + total) Y 1.000]. Absolutevalues for cAMP accumulation varied between experiments,but variability within a given experiment was in general<10%.

Membrane protein preparation andimmunodetection of a,, expressionHEK293 cells were grown on I 50-mm-diameter dishes to

70—80% confluence. Transfection was performed as in 12-well plates with proper adjustments to the volumes andamounts of the reagents used. Transfected cells were incu-

J. Neuroehen,.. Vol. 65, No. 6, 1997

2516 M. K. C. HO AND Y. H. WONG

bated with growth media at normal growth conditions for48 h for the expression of exogenous proteins. Cells on eachplate were then washed with Ca2~/Mg2~-freephosphate-buffered saline and harvested with 5 ml of Ca2~/Mg2~-free phosphate-buffered saline containing 10 mM EDTA.All subsequent steps were performed at 4°C.After pelleting,cells were resuspended in hypotonie lysis buffer (50 mMTris-HCI, 2.5 mMMgC1

2, I mMEGTA, I mMphenylmeth-ylsulfonyl fluoride, 1 mM benzamidine-HCI, and 1 mM di-thiothreitol, pH 7.4) and lysedby one cycle offreeze—thaw-ing followed by 10 passages through a 27-gauge needle.Nuclei were removed by low-~peedcentrifugation (200 g,5 mm), and membranes were collected by spinning the su-pernatants at 15,000 g for 15 min. The pellets were finallyresuspended in ly.sis buffer. Protein concentrations were de-termined using the Bio-Rad Protein Assay Kit. For eachsample, 50 p.g of membrane proteins was separated on a12.5% sodium dodecyl sulfate-polyacrylamide gel and dee-trophoretically transferred to nitrocellulose membranes. Pro-tein markers on the membrane were localized by Ponceau Sstaining. Immunodetection of a, by the a,-specifie antiserump-961 (Casey et al., 1990) was visualized by ehemilumines-cence using the ECL kit from Amersham Corp.

Trypsin resistance assayTrypsiniztttion of a, subunits was performed as described

previously (Berlot and Boume, 1992) with minor modifica-tions. Membrane proteins (150 pg) from transfectedHEK293 cells were solubilized in 120 p,

1 of ice-cold 20mMHEPES (pH 8.0) containing 10 mM MgCl

2, 1 mM EDTA,2 mM fi-mercaptoethanol, and 0.64% (wt/vol) Lubrol PXfor 18—20 h with gentle shaking at 4°C.Solubilized mem-brane proteins were collected by centrifugation at 13,000rpm for 5 min. The samples were incubated at 30°Cfor 30min in the absence or presence of 150 p.M guanosine S‘-O-(3-thiotriphosphate) (GTPyS). Where indicated, N-tosyl-L-phenylalanine ehloromethyl ketone-treated trypsin wasfreshly diluted with solubilization buffer and added to thesamples at a final concentration of 5 pg/mI, andthe mixtureswere incubated at 30°Cfor 5 min. The reaction was termi-nated by adding soybean trypsin inhibitor to a final concen-tration of I mg/ml. The samples were then immunoblottedwith the a,-speeific antiserum 3A-l70 (Gramseh Laboratory,Schwabhausen, Germany) as described above.

RESULTS

Manning and co-workers generated three differentphosphorylation-resistant mutant a, subunits by site-directed mutagenesis (Lounsbury et al., 1993). Herewe made use of these phosphorylation-resistant a, mu-tants to compare their signaling properties with thoseof a,WT. The a, polypeptide has two PKC-phosphory-lation sites at the N-terminal serine residues numbered16 and 27. The three a, mutants have one or moreof the serine residues changed into alanine, hereafterdenoted as a, 16, a,27, and a, 16/27, of which the num-bers indicate the mutation sites (Fig. 1). a,l6 anda,27 have either one of the two phosphorylation sitesmutated, and the proteins expressed in HEK293 cellscan be phosphorylated at the unchanged sites, whereasa,16/27 is completely resistant to PKC-mediatedphosphorylation (Lounsbury et al., 1993).

FIG. 1. Schematic diagram of the N-terminal sequences of wild-type and phosphorylation-resistant mutants of a,. Sequences ofthe first 30 amino acids of a,, a,16, a,27, and a,16/27 are de-picted. The solid bar indicates the putative receptor couplingdomain of a,. The hatched bar maps the putative ßy-bindingregion of a,. Myristate and palmitate are incorporated to G1y

2(A) and Cys‘ (U), respectively. The amino acid residues thatare presumably in direct contact with the /3 subunit are marked(•). Numbers at the bottom indicate relative positions of theresidues.

When coexpressed with a, in HEK293 cells, G,-coupled receptors, such as the dopamine D

2 receptor(D7R), adenosine A1 receptor [A1R (Wong et al.,1992)], and 6-opioid receptor [ÖOR (Tsu et al.,1995a)], can perform PTX-insensitive inhibition ofintracellular cAMP levels on the addition of appro-priate agonists. We used a similar approach to assessthe signaling properties of the phosphorylation-resis-tant mutants of a,. Cells were transfected with cDNAsencoding a5QL [a constitutively active G5a subunit(a5) mutantfor elevating the basal activities of endoge-nous ACs (Masters et al., 1989)], ÔOR, or D2R andone of the four a, subunits. Transfected cells werelabeled with [

3H]adenine for monitoring the changeof cAMP levels, and PTX was added at the same timeto uncouple G

1/G,, proteins from the receptors. As ex-pected, a,-transfected cells showed significant inhibi-tion of a,QL-stimulated endogenous AC activity whenthey were subjected to an acute treatment with 100n M [D-Pen

2,D-Pen5] enkephalin (DPDPE: 6-selectiveagonist) or 10 pM quinpirole (D

2-selective agonist;Fig. 2). Such a response was specifically mediated viaa, because endogenous G~/G,,proteins were uncoupledby PTX. Control experiments using cells transfectedwith a~2(or pcDNAI) failed to give significant inhibi-tion of cAMP levels (Fig. 2). Under similar experi-mental conditions, all three phosphorylation-resistanta, mutants behaved just like a,. Comparable extentsof reductions of cAMP levels were observed in thecells coexpressing any one of the mutants (Fig. 2).These results indicate that all three a, mutants wereable to interact with the 6OR and the D,R and inhibitendogenous AC in a manner identical to that of a,.

Among the different isozymes of AC, the type VIisozyme (AC6) has been shown to be more sensitiveto the inhibitory effect of a1,. (Chen and Iyengar, 1993;Taussig et al., 1993). We coexpressed AC6with a5QL,6OR, or D2R and one of the four a, subunits in anattempt to enhance the receptor-induced inhibitory sig-

‘a

J. N,‘uro,.‘he,,i., Vol. 68, No. 6, 1997

ROLE OF SER‘6 AND SER27 IN Ga,. FUNCTIONS 2517

FiG. 2. PTX-insensitive inhibition ofcAMP accumulation by a, and itsphosphorylation-resistant mutants.HEK293 cells were cotransfectedwith an inhibitory G protein a sub-unit(aj

2, a,, a,16, a,27, ora,16/27),at 0.03 pg/mI, a5QL (0.15 pg/mI),or ÖOR or D2R (0.25 pg/mI) asindicated. Transfected cells weretreated with PTX (100 ng/ml) andlabeled with [

3H]adenine (1 pCi/ml). The cAMP accumulation wasassayed in the absence or presenceof agonists (100 nM DPDPE for bORor 10 pM quinpirole for D,.R) with 1mM IBMX. The cAMP levels weremeasured as the ratios of radioactivity of cAMP-containing frac-tions to that of the corresponding total labeled nucleotide frac-tions. Results are expressed as percent inhibition of the basalvalues (as defined in Materials and Methods), which ranged from1.52 © 0.29 to 2.05 ±0.22 for bOR-transfected cells and from4.39 ±0.53 to 5.29 ±0.74 for D,.R-transfected cells. Data aremean ±SD (bars) values of triplicate trials in one single experi-ment. *p <~ 0.05 by paired t test, agonist-induced reduction ofcAMP levels significantly greater than that of cs,.-transfectedcells. Paired t test analysis of results obtained from four indepen-dent experiments indicated that the differences between the re-sponses of each mutant a, subunit and a,WT were insignificant.

nais. As shown in Fig. 3, DPDPE or quinpirole treat-ment produced a prominent a,-mediated inhibitory ef-fect in cells eoexpressing AC6 (cf. Fig. 2). Similarenhancements of inhibitory effects were observed incells coexpressing the three a, mutants. These resultsimplied that the point mutations introduced at the N-terminus of a, had no obvious effect on its ability toinhibit AC6 or endogenous ACs in HEK293 cells. TheIwo N-terminal serine residues of a, did not appear toplay a critical role in effector coupling.

Many lines of evidence support the importance ofthe N-terminus of the G protein a chain on subunitinteraction. A recent in vitro study reported that phos-

~ihorylated a, subunit bound fly subunits with much

FIG. 3. Inhibition of AC6 by variousa, subunits. HEK293 cells weretransfected, PTX-treated, labeled,and assayed for cAMP accumula-tion as in the legend to Fig. 2 except0.25 pg/mI AC6 was added duringtransfection. Agonist-induced inhi-bition of a

5QL-stimulated AC6 activ-ities was expressed as percent inhi-bition of the basal values, whichranged from 6.87 ± 0.65 to 10.10‚- 0.65 for 50R-transfected cellsand from 9.31 ± 1.57 to 12.93~ 0.99 for D2R-transfected cells.Data are mean ±SD (bars) values oftriplicate determinations in a singleexperiment. *p < 0.05 by paired t test, inhibitory responsesmediated by the four a, subunits significantly greater than thatof control cells. From three separate experiments, no significantdifference between mutant a,- and a,WT-mediated responseswas detected with paired t test.

weaker affinity than the unphosphorylated form (Fieldsand Casey, 1995). The reassociation of G protein tri-mers was presumably prevented by PKC-mediatedphosphorylation (Fields and Casey, 1995). However,initial reports on the phosphorylation of G, protein(Lounsbury et al., 1991, 1993) suggested that the a,subunit can be phosphorylated even as a trimer withfly subunits. We made use of the three a, mutants toinvestigate whether the two serine residues concernedwere important per se for subunit dissociation on re-ceptor activation. Functional release of fly subunitsfrom activated a, was monitored by using the fly-sensitive type II AC (AC2). This isozyme has beenshown to be synergistically stimulated by G proteinfly subunits and GTP-bound a5 (Lustig et al., 1993)and is less sensitive to a1- and a,-mediated inhibition.By coexpressing AC2 with a5QL, ÔOR, and a,, theAC2 activity was significantly stimulated by additionof DPDPE even after PTX treatment (Fig. 4, upperpanel). Comparable levels of stimulation were alsoobserved in cells transfected with one of the three a,mutants and by using different receptors (D2R or AIR)and agonists [quinpirole or R ( — ) - N

6- (2-phenyliso-propyl)adenosine] to substitute for 60R and DPDPE,respectively (Fig. 4, middle and lower panels). It sug-gested that all of these a, mutants could release suffi-cient amounts of fly subunits for potentiating a

5QL-stimulated AC2. The results showed that the point mu-tations did not impair the functional release of flysubunits from a,. Thus, the two serine residues maynot be important in subunit dissociation.A previous study has demonstrated that N-terminal

mutations can affect the ability of a, to inhibit AC

FIG. 4. Stimulation of AC2 by /Jysubunits released from various a,subunits. HEK293 cells were co-transfected with aeQL (0.025 pg/ml), AC2 (0.25 pg/mI), one of threereceptors (50R, D2R, or A1R; 0.25pg/mi), and inhibitory a subunits(a2, a,, a,16, a,27,ora,16/27; 0.03pg/mi). Transfected cells werePTX-treated and labeled with [

3H]-adenine. The cAMP accumulationwas assayed in the absence orpresence of the appropriate ago-nists [100 nM DPDPE for 50R, 10p.M quinpirole for D,.R, or 10 p.MR( —) -N6-(2 - phenylisopropyl)ade-nosine for A

1RI with IBMX. Resultsare expressed as percent re-sponses over the basal values,which ranged from 2.52 ±0.05 to4.36 ± 0.35 for bOR-transfectedcells, from 2.48 ±0.21 to 2.77 ±0.20 for D2R-transfected cells,and from 2.02 ±0.17 to 3.78 ±0.59 for A1R-transfected cells.Data are mean ±SD (bars) values of triplicate trials in a singleexperiment. *p <0.05 by paired t test, agonist-induced increaseof cAMP levels significantly greater than that of control cells.Similar results were obtained in five independent experiments,and the mutant a,- and a,WT-mediated responseswere not sig-nificantly different by paired t test.

J. Neuroche,n., Vol. 68, No. 6, 1997

2518 M. K. C. HO AND Y. H. WONG

constitutively. G~-mediatedinhibition of AC is en-hanced by an N-terminal mutation that prevents palmi-toylation of a, (Wilson and Boume, 1995). TheGTPase-deflcient mutant of a, [a,Q205L (Wong etal., 1992)] exhibits slightly impaired inhibitory controlon AC when its N-terminal fatty acylation sites (Gly2and Cys3) are mutated (Wilson and Boume, 1995).Thus, we sought to examine whether mutations at Ser‘6and Ser27 would alter a,‘s ability to achieve the activeconformation by introducing a second mutation at co-don 205 (GIn replacedby Leu). Constitutive inhibitionof AC by the resultant GTPase-deficient mutants wasexamined in transfected HEK293 cells. Cells were co-transfected with the luteinizing hormone receptor(LHR) and one of the QL mutants or their correspond-ing wild types. Inclusion of LHR allowed us to stimu-late cAMP accumulation in transfected cells with 10ng/ml human choriogonadotropin (hCG). When com-pared with their corresponding wild-type constructs,the QL mutants of a,, a,16, and a,27 significantlyreduced the hCG-stimulated cAMP levels (Fig. SA).lt is interesting that constitutive inhibition of the cAMPlevel was not observed in a,16/27QL-transfected cells(Fig. SA).

The apparent loss-of-function phenotype of a,16/27QL prompted us to examine the constitutive inhibi-tory activities of the QL mutants in detail. HEK293cells were cotransfected with LHR and increasing con-centrations of the various QL mutants. For cells ex-pressing a,QL, a,16QL, and a,27QL, the hCG-stimu-lated cAMP accumulations were significantly attenu-ated at eDNA concentrations of ~0.03 ‚ug/ml, andmaximal reductions were observed at or around 0. I

FIG. 5. GTPase-deficient a,1 6/270L lost the constitutive inhibitoryeffect on AC. A: HEK293 cells werecotransfected with [HR(0.15 pg/ml) and either wild-type (WT)or GTPase-deficient (QL) mutantforms of a,, a,16, a,27, or a,1 6/27.[‘H I Adenine-labeled transfectedcells were stimulated with hCG (10ng/ml) to elevate the cAMP levels.Results are expressed as percentresponse of hCG-stimulated cAMPlevels obtained in control cellstransfected with [HR and pcDNAivector (0.3 pg/mI). The hCG-stim-ulated cAMP levels ranged from2.35 ± 0.30 to 3.26 ± 0.37. B:HEK293 cells were cotransfectedwith [HR and increasing concen-trations of one of the four GTPase-deficient a, mutants. Totalamounts of DNA transfected were maintained at 0.45 pg/mI byadding pcDNA1 vector. Cells were labeled and assayed for cAMPaccumulation as in A. Results are expressedas percent responseof the hCG-stimulated cAMP levels of cells transfected in theabsence of a, subunit and ranged from 9.63 ±0.77 to 10.41±1.04. Data are mean ±SD (bars) values of triplicate trials inone singleexperiment. Threeseparate experiments yielded simi-lar results. *p < 0.05 by paired t test, significantly different froma,Q[.

FIG. 6. a,16/27Q[ can neitherscavenge ßy subunits nor coupleto ‚5OR. HEK293 cells were cotrans-fected with a~Q[ (0.025 pg/mI),AC2 (0.25 pg/mI), ÔOR (0.25 pg/ml), and pcDNAI vector, transducin(a,), a,WT, or a,16/27Q[. Trans-fected cells were labeled with [3H]-adenine with (lower panel) or with-out (upper panel) PIX. The cAMPaccumulation was assayed in theabsence or presence of 100 nMDPDPE with IBMX. Data are mean±SD (bars) values of triplicate tri-als in a single experiment. Similarresults were obtained in three sep-arate experiments. *p < 0.05 by paired t test, significant in-creases of cAMP levels when compared with the correspondingbasal values.

‚ug/ml eDNA (Fig. SB). In contrast, the hCG-inducedelevation of intracellular cAMP level was only mod-estly affected in cells expressing a,16/27QL. Even atthe highest concentration of eDNA used, the cAMPlevel was inhibited by 30% only (Fig. SB). Simultane-ous replacement of Ser‘6 and Ser27 by Ala appeared toperturb the GTP-induced active conformation of a,l6/27QL, leading to impairment of its ability to inhibitAC constitutively.

Because the N-terminal point mutations were lo-cated in the putative fly-binding domain, it is conceiv-able that a,16/27QL may exhibit altered affinity forfly subunits. We addressed this possibility indirectlyby testing the ability of a,16/27QL to release or scav-enge fly subunits in the AC2 system. HEK293 cellswere cotransfected with AC2, 60R, and a,QL in theabsence or presence of a,I6/27QL or ai. WithoutPTX treatment, addition of DPDPE led to a significantincrease of cAMP level, which was attributed to therelease of fly subunits via activation of endogenousG

1 proteins. Coexpression with a,1 abolished the fly-mediated effect because a,1 is an efficient fly scaven-ger (Fig. 6). However, fly-mediated stimulation ofAC2 was unaffected in cells cotransfected with a,l6/27QL (Fig. 6), implying that a,16/27QL could notremove the fly subunits released from G1. Unlike a,,a,16/27QL did not provide PTX resistance to DPDPE-induced stimulation of AC2 in the transfected cells(Fig. 6), indicating a lack of interaction with ÔORand fly subunits. Thus, the loss of the constitutiveinhibitory effect of a, I 6/27QL was probably not dueto steric hindrance caused by the binding of fly sub-units. a,l6/27QL appeared to remain in a fly-free butinactive form.

We further examined whether the different a, con-structs could be expressed to comparable levels inHEK293 cells when other recombinant proteins werecoexpressed simultaneously. Figure 7 shows the immu-nodetection of the different a, subunits in crude mem-brane preparations obtained from cells eoexpressingthe ÖOR, AC2, a5QL, and one of the a, subunits. Con-

j. Neurochein., Vol. 68, No. 6, 1997

ROLE OF SER‘6 AND SER27 IN Ga,. FUNCTIONS 2519

FIG. 7. Expression of recombinant a, subunits in HEK293 cells.HEK293 cells were cotransfected with ÖOR (0.25 pg/mI), AC2(0.25 pg/mI), a

5Q[ (0.025 pg/mI), and pcDNAI vector, a,, a,16,a,27, or a,16/

27 (0.03 pg/mI). Upper panel: Membrane pro-teins from transfected cells were separated by sodium dodecylsulfate—polyacrylamide gel electrophoresis and transferred toa nitrocellulose sheet for immunodetection with the a,-specificantiserum P-961 (WT). Lower panel: Results from a similar ex-periment using the four GTPase-deficient mutants (QL). Controlcells transfected with pcDNAI showed no detectable signal. Twoindependent experiments using different batches of membraneproteins yielded similar results.

trol cells expressing all of the above recombinant pro-teins except a, showed no detectable signal (Fig. 7,pcDNAI). Detection of recombinant wild-type a,,a,16. a,27, and a,16/27 by the a,-specific antiserumP-961 (Casey et al., 1990) was specific, and their lev-els of expression were comparable (Fig. 7, upperpanel). Similar results were obtained for those a, sub-units harboring the Q2OSL mutation (Fig. 7, lowerpanel). Hence, the lack of constitutive suppression ofAC by a,16/27QL was not due to poor expression ofthat a, mutant.

Finally, to confirm that a,16/27QL can indeed adopta GTP-bound activeform, we determined its sensitivityto tryptic digestion. GTP- or GTPyS-bound a subunitsare known to be protected from cleavage at a conservedarginine residue by trypsin. Purified a, has been shownto be trypsinized to small fragments undetectable bysodium dodecyl sulfate—polyacrylamide gel electro-phoresis and western blot, whereas GTPyS-bound a,W~Scleaved to a slightly smaller product (~-~37kDa)by trypsin (Fields and Casey, 1995). If a,16/27QL isconstitutively active, it should be resistant to trypsin-ization even in the absence of GTPyS. Membrane pro-teins of HEK293 cells expressing either a,QL or a,l6/27QL were solubilized by the nonionic detergent Lu-brol PX and subjected to limited trypsinization in theabsence or presence of ISO ‚uM GTPyS (Fig. 8). Botha,QL and a,I 6/27QL were only partially digested bytrypsin in the absence of GTPyS. A similar proteolyticfragment was detected in a,QL and a,16/27QL sam-

111es containing GTPyS. In the absence of GTPyS,a,WT and a,16/27 were digested to undetectable frag-ment sizes (Fig. 8). These results indicate that, likers,QL, a,16/27QL was able to remain in the GTP-hound active conformation.

DISCUSSION

Numerous biochemical sign posts point to the poten-tial importance of the N-terminal region of the G pro-

tein a subunits in regulating receptor and fly couplingevents. In the case of G,, two serine residues that serveas PKC-mediated phosphorylation sites are locatedwithin this region. This prompted us to explore thefunctional significance of these serine residues. De-tailed biochemical characterization of the three a, mu-tants used in the present study was previously reported(Lounsbury et al., 1993). By adopting the same mam-malian expression system, we showed that these serineresidues in a, are not absolutely required for receptorrecognition, effector coupling, and fly binding. Theloss of PKC-targeted serine residues of a, did not dras-tically affect its ability to transduce signals from threedistinct receptors (ÖOR, D

2R, and A1R) to three differ-ent AC systems (endogenous AC, AC2, and AC6).

Preservation of receptor recognition by the 5er ~andSer

27 mutants of a, is not entirely unexpected. It isgenerally assumed that the critical determinants forreceptor coupling are confined in the C-terminus ofthe a subunit. Direct involvement of the N-terminusof the a subunit in receptor binding is less apparent.Nevertheless, the N-terminus may indirectly contributeto receptor coupling by either stabilizing the essentialreceptorbinding domain at the C-terminus or recruitingadditional receptor binding surfaces lying on fly sub-units. Indeed, the activation of G

0 by mastoparan canbe abolished by site-directed mutagenesis (Denker etal., 1992) or tryptic removal (Denker et al., 1995) ofthe N-terminal amino acids of a0, and a peptide derivedfrom residues 8—23 of transducin has been shown toblock the rhodopsin—transducin interaction (Dratz etal., 1993). Recent analysis of the crystal structures ofmonomeric GDP-bound a11 revealed that both terminiare apparently associated with each other (Mixon etal., 1995). A structurally identifiable domain mightbe created by the two associated termini to facilitatereceptor recognition. Even if this microdomain existsin a,, the C-terminal residues may contribute moretoward defining the specificity of receptor coupling.Indeed, the last five amino acids of a, have been shownto be sufficient in allowing the D2R to interact produc-

FIG. 8. Trypsin sensitivity of a,16/27Q[. HEK293 cells weretransfected with a,, a,16/27, a,Q[, or a,16/27Q[. Membraneswere prepared from the transfected cells, treated with trypsin,resolved on 10% sodium dodecyl sulfate—polyacrylamide gelelectrophoresis, and immunoblotted with the a,-specific antise-rum 3A-1 70. The first lane in each set is the control (no trypsin).The second and third lanes show the results of trypsinization.Where indicated, 150 pM GTP7S was used to shift the a~sub-units to the trypsin-resistant active conformation. Two indepen-dent experiments yielded similar results.

J. Neurochen,., Vol. 68, No. 6, 1997

2520 M. K. C. HO AND Y H. WONG

tively with the G,1 a subunit (a,1) (Conklin et al., 1993)and aI2 (Voyno-Yasenetskaya et al., 1994).

Much less is known with respect to the effector-interacting domain on a,. AC isozymes represent theonly effectors of G, identified to date. Studies on othera subunits showed that the essential effector couplingdomains may reside on the C-terminal half of the poly-peptide (Berlot and Boume, 1992; Venkatakrishnanand Exton, 1996). However, it is unclear if cognateregions on a, are involved in effector coupling and, ifso, whether additional domains are required. There areprecedents to suggest that the N-terminus of an a chainmay affect the efficacy of effector coupling. N-termi-nus-truncated a, exhibits impaired ability to activateAC and does not bind fly subunits (Joumnot et al.,1991). Similarly, N-terminus-trypsinized aq or its pal-mitate-deficient mutant is unable to activate phospholi-pase Cfi1 (Hepler et al., 1996). Myristate- and palmi-tate-deficient mutants of a, exhibit altered ability toregulate ACs (Wilson and Boume, 1995). Althoughreduced membrane attachment of these fatty acylation-deficient a, mutants might account for impairment ofcoupling to AC, the enhanced ability of the palmitate-deficient mutant of a, to inhibit AC (Wilson andBoume, 1995) tends to indicate that the N-terminus ofa, may contribute to effector regulation. Our resultssuggested that the two PKC-targeted serine residuesof a, might not play a critical role in the inhibition ofAC. All four a, isoforms inhibited endogenous ACsand, more prominently, recombinant AC6 in HEK293cells at similar efficacies. The two point mutations in-troduced at the N-terminal serine residues of a, did notappear to affect its efficacy or specificity for regulatingACs. This is in agreement with a recent reconstitutionstudy using recombinant proteins, where PKC-medi-ated phosphorylation of a, had little or no effect onits inhibitory interactions with type V AC (Kozasa andGilman, 1996).

fly-Mediated AC2 stimulation is a convenient andconsistent functional assay to test the efficiency of re-leasing fly subunits from a G protein trimer. Throughthe activation of OOR, D2R, or A1R, fly subunits werereleased from a,WT as well as from the three a, mu-tants and stimulated AC2 to comparable extents. Theefficiency of releasing fly subunits was not affectedby the point mutations at Ser

16 and Scm27 of a,, whosecognate residues in a,

1 and a11 form part of the flybinding surface (Wall et aI., 1995; Lambright et al.,1996). The oxygen atom of the hydroxyl group of5er‘

2 of transducin (corresponding to 5er‘6 of a,) ispredicted to form a hydrogen bond with the amidicnitrogen atom of Lys8‘2 of the fi, subunit. However,Lys°‘of fi

1 appears to interact with three differentamino acid side chains on the N-terminal helix of a,1(Sen

2, Leu‘5, and GIn‘6). lt is entirely conceivablethat the loss of Semis of a, may not hinder its associa-tion with the fi subunit. The residue on a,

1 correspond-ing to 5er

27 of a, is actually Ala23, which associateswith Leu55 of fi, through hydrophobic van der Waals

forces (Lambright et al., 1996). Hence, substitution ofScm27 of a, by alanine should not severely perturb theintermolecular interactions between a, and the fi sub-unit. Direct comparison between a,, and a, is probablyvalid because the relative orientations of the aminoacid side chains on the N-terminal a-helices are almostidentical. Both Scm‘6 and 5er27 of a, are presumablyfacing the fi propeller and may interact with the fisubunit through intermolecular forces that are similarto those described for a,,.

The loss-of-function phenotype of the triple mutant,a,16/27QL, was somewhat surprising because a,l6/27 behaved just like a, in terms of both inhibition ofAC and fly release. GIn204 of a,i is known to stabilizethe transition state of GTP during hydrolysis (Colemanet al., 1994), and loss of this residue abolishes GTPaseactivity without altering the steps from GDP dissocia-tion to GTP binding. The cognate mutation at GIn2“5 ofa,16/27 should produce a eonstitutively active mutant.One possible scenario is that a,16/27QL might not beproperly anchored to the membrane and hence cannotinhibit AC. It is conceivable that the 5er mutationsmight weaken a,‘s association with fly, but not to theextent that receptor-mediated signaling is lost. Intro-duction of the GTPase-deficient mutation might furtherweaken the fly binding so that the resultant a,l6/27QLis loosely attached to the plasma membrane and couldnot inhibit AC. However, the amount of a,16/27QLpresent in the plasma membrane was not significantlylower than that of the other a, constructs, be it wild-type or mutant forms (Fig. 7).

Another provocative explanation for the observedphenotype of a,16/27QL is that mutations of 5er‘6and 5er27 to Ala actually produced an a, subunit thattightly binds the fly subunit, despite the presence ofthe GIn205 mutation. It is noteworthy that 5er27 is cog-nate to Ala23 of a,

1 and that the transducins are re-nowned for their high affinity for fly. Binding of flyto the N-terminus of a,16/27QL might occlude theputative effector-binding surfaces on the N-terminus(Hepler et al., 1996), switch II (where Gln

211~is situ-ated), and surrounding regions (Bemlot and Boume,1992; Venkatakrishnan and Exton, 1996) from inter-acting with AC. However, specific interactions be-tween the switch II helix and fly are likely to be af-fected by the QL mutation as GIn205 forms part of thefly-contact surface (Lambright et al., 1996). Confom-mational changes of switch II have been linked to re-ceptor interactions (Mixon et al., 1995). In pseudohy-poparathymoidism, a mutation in the switch II regionof a, has been shown to impair receptor stimulation(Farfel et al., 1996). Our tmypsin resistance experimentclearly showed that a,l6/27QL can indeed adopt theGTP-bound conformation and thus is unlikely to betightly associated with the fly complex. As yet, we donot have an adequate explanation for the a,16/27QLphenotype because several questions remain unre-solved. These questions pertain to whether the N-ter-minal serine mutations alter the rate of GDP dissocia-

.1. N,‘,,r,,cl,,‘m., Vol. 68, No. 6. /997

ROLE OF SER‘6 AND SER27 IN Ga,. FUNCTIONS 2521

lion, the duration of an “empty“ state, or the mate ofGTP-induced conformational change.

Scm‘6 (as well as the surrounding residues) is con-served within the a, subfamily [a,, a,, G

0a subunit(a,,), and a1 subtypes], but Scm

27 is unique for a,.Phosphorylation of these serine residues by PKC canseemingly prevent subunit meassociation of G, (Fieldsand Casey, 1995). Given that both serine residues are

l)rcdlcted to participate in thebinding of fly, the attach-nient of bulky phosphate moieties may severely disruptintermolecular forces required for association with the

13 subunit. For instance, the mere presence of a phos-phate group may prevent the formation of ion pairs inihe vicinity of the serines (Asp21‘ and Glu26 of a, pair-ing with Lys5‘ and Leu55 of fi,, respectively). Therelative importance of phosphorylating 5er 6 and 5er77with regard to G, signaling process remains to be deter-mined. Because a,16, a,27, and a,16/27 did not ex-hihit altered abilities in receptor coupling, subunit dis-sociation, and effector interaction, they represent use-ful tools in assessing the importance of 5er‘6 and 5er27in a, signaling on PKC-mediated phosphorylation. Re-examination of the signaling properties of wild-typeand mutant forms of a, in the presence of phorbolesters may yield different results. However, it shouldhe noted that PKC-mediated phosphorylation did notappear to impair the ability of recombinant a, to inhibittype V AC (Kozasa andGilman, 1996) and that endog-enous phosphomylation of a, can occur in HEK293cells (Lounsbury et al., 1993). The complexity of theG protein-mediated signaling network is almost be-yond comprehension. One can easily envisage a phos-

phorylation-dependent ‘‘shut off“ or “turn on“ mech-anism for a, that may provide a way to divert signalsalong specific routes.

Acknowledgment: We are very much indebted to thefollowing persons for their generous donations: David Man-ning for the wild-type and mutant human a, eDNA clones,Patrick Casey for the a,-specifie antiserum P-961, Ravi Iyen-gar for AC6 eDNA, Randall Reed for AC2 eDNA, and ChrisEvans for the mouse ÔOR eDNA. We thank Henry Boumetor inspirational discussions and Joy Chan and Lisa Yungtor technical assistance. This work was supported in part bygrants HKUST 169/93M and HKUST 567/95M from theResearch Grants Council of Hong Kong.

REFERENCES

Animer H. and Schulz R. (1994) Retinoic acid-induced differentia-tion of human neuroblastoma SH-SY5Y cells is associated withchanges in the abundance of G proteins. J. Neurochern. 62,1310—1318.

IterIol C. H. and Boume H. R. (1992) identification of effector-activating residues of G,,,. Cell 68, 911—922.

(‘arlson K. E., Brass L. F., and Manning D. R. (1989) Thrombinand phorbol esters cause the selective phosphorylation of a Gprotein other than G, in human platelets. J. Biol. Chem. 264,13298—l 3305.

(‘asey P. J., Fong H. K. W., Simon M. I., and Gilman A. G. (1990)G,, a guanine nucleotide-binding protein with unique bioehemi-cal properties. J. Biol. Chew. 265, 2383—2390.

Chen J. and lyengar R. ( 1993) Inhibition ofeloned adenylyl cyclasesby mutant-activated G,, and specific suppression of type 2 ade-nylyl cyclase inhibition by phorbol ester treatment, J. hot.Chew. 268, 12253--l2256.

Coleman D. E., Berghuis A. M., Lee E., Linder M. E., Gilrnar, A. G..and Sprang S. R. (1994) Structures of active conformations ofG,,, and the mechanism of GTP hydrolysis. Science 265, 1405—1412.

Conklin B. R., Fand Z., Lustig K. D., Julius D., and Boume H. R.(1993) Substitution of three amino acids switches receptorspecificity of G,

1a to that of G1a. Nature 363, 274—276.Denker B. M., Neer E. J., and Schmidt C. J. (1992) Mutagenesis ot

the amino terminus of the a subunit of the G protein G,,. Invitro characterization of‘ a,,ßy interactions. .1. Blc,l. Cl,eo,. 267,6272—6277.

Denker B. M., Boutin P. M., and Neer E. J. (1995) interactions be-tween the amino- and carhoxyl-terminal regions of Ga subunits:analysis of mutated Ga/Ga17 chimeras. Biochemistry 34,5544—555 3.

Dratz E. A., Furstenau J. E., Lambert C. G., Thi,‘eaolt D. L., RarickH., Schepers T.. Pakhlevaniants S., and Hamm H. E. (1993)NMR structure of a receptor-bound G-protein peptide. Nature363, 276—281.

Farfel Z., lin T., Shapira H., Roitman A., Mouallem M.. and BoumeH. R. (1996) Pseudohypoparathyroidisni, a novel mutation inthe ßy-contact region of G,a impairs receptor stimulation. J.Biol. C/mm. 271, 19653—19655.

Fields T. A. and Casey P. J. (1995) Phosphorylation of G,,, by pro-tein kinase C blocks interaction with the ßy complex. J. Blot.Chew. 270, 23119—23125.

Fong H. K. W., Yoshimoto K. K.. Eversole-Cire P., and Simon M. I.(1988) identitication of a GTP-binding proteiil alpha subunitthat lacks an apparent ADP-ribosylation site for pertussis toxin.Proc. Nati. Acod.Sci. USA 85, 3066—307t).

1-lallak H., Muszhek L., Laposata M., Belmonte E.. Brass L. F., andManning D. R. (1994) Covalent binding of arachidonate to Gprotein a subunits of human platelets. J. Blot. Chew. 269,4713—4716.

Hepler J. R., Biddlecome G. I-l., Kleuss C.. Camp L. A., HofmannS. L., Ross E. M., and Gilman A. G. (1996) Functional impor-tance of the amino terminus of G,,,,. J. Blot. Chew. 271, 496—504.

Journot L., Pantaloni C., Boekaert J., and Audigier Y. (1991) Dele-tion within the amino-terminal region of G,,, impairs its abilityto interact with ßy subunits and to activate adenylate cyclase.J. Blot. Chew. 266, 9009—9015.

Kozasa T. and Gilman A. G. (1995) Purification of recombinant Gproteins from Sf9 cells by hexahistidine tagging of associatedsubunits. Characterization of a1, and inhibition of adenylyl cy-clase by a,. J. Blot. Chew. 270, 1734—1741.

Kozasa T. and Gilman A. G. (1996) Protein kinase C phosphorylatesG2,, and inhibits its interaction with G23,. J. Blot. Chew. 271,12562—12567.

Lai H. W. L., Minami M.. Satoh M., and Wong Y. H. (19951 G,coupling to the rat K-opioid receptor. FEBS Let!. 360, 97—99.

Lambright D. G., Sondek J.. Bohm A., Skiha N. P., Hamm H. E.,and Sigler P. B. (1996) The 2.0 A crystal structure of a hetero-trimeric G protein. Nature 379, 311 —319.

Linder M. E., Middleton P., Hepler J. R.. Taussig R.. Gilman A. G..and Mumby S. M. f 1993) Lipid modifications of G proteins:alpha subunits are palmitoylated. Proc. Na/I. Aca,t.Sci. USA

90, 3675—3679.Lounsbury K. M., Casey P. J., Brass L. F., and Manning D. R.

(1991) Phosphorylation of G, in human platelets. Selectivityand site of modification. J. Blot. Cheo,. 266, 22051 —22056.

Lounsbury K. M., Schlegel B., Poncz M., Brass L. F.. and ManningD. R. (1993) Analysis of G,,, by site-directed mutagenesis. Sitesand specificity of protein kinase C-dependent phosphorylation.J. Blot. Chein. 268, 3494—3498.

Lustig K. D.. Conklin B. R., Herzmark P., Taussig R., and BoumeH. R. (1993) Type Il adenylyl cyclase integrates coincidentsignals from G,, G1. and G,~.1. Blot. Che,n. 268, 13900—139(15.

.1. Ne,,roch,‘,,,.. Vol. 68, No. 6, /997

2522 M. K C. HO AND Y H. WONG

Masters S. B., Miller R. T., Chi M. H., Chang F-H., Beiderman B.,Lopez N. G., and Boume H. B. (1989) Mutations in the GTP-binding site of G,,, alter stimulation of adenylyl cyclase. J. Blot.Chew. 264, 15467—15474.

Matsuoka M., Itoh H., Kozasa T., and Kaziro Y. (1988) Sequenceanalysis of eDNA and genomic DNA for a putative pertussistoxin-insensitive guanine nucleotide-binding regulatory proteina subunit. Proc. Nail. Acad. Sei. USA 85, 5384—5388.

Mixon M. B., Lee E., Coleman D. E., Berghuis A. M., Gilman A. G.,and Sprang S. R. (1995) Tertiary and quaternary structuralchanges in G,,

1 induced by GTP hydrolysis. Science 270, 954—960.

Mumby S. M., Heukeroth R. O., Gordon J. I., and Gilman A. G.(1990) G-protein alpha-subunit expression, mynistoylation, andmembrane association in COS cells. Proc. Nail. Acad. Sei. USA87, 728—732.

Shum J. K., Allen R. A., and Wang Y. H. (1995)The human chemo-attractant complement C5a receptor inhibits cyclic AMP accu-mulation through G, and G, proteins. Biochem. Biophy.c. Res.Commun. 208, 223—229.

Taussig R., iniguez-Lluhi J. A., and Gilman A. G. (1993) Inhibitionof adenylyl cyclase by G~,,.Science 261, 218—221.

Tsu R. C., Chan J. S. C., and Wong Y. H. (l995a) Regulation ofmultiple effectors by the cloned ö-opioid receptor: stimulationofphospholipase C and type II adenylyl cyclase. J. Neurochem.64, 2700—2707.

Tsu R.C., Lai H.W.L., Allen RA., and Wong Y.H. (1995h)Differential coupling of the fonnyl peptide receptor to adenylate

cyclase and phospholipase C by the pertussis toxin-insensitiveG, protein. Biocheo,. J. 309, 331—339.

Venkataknishnan G. and Exton J. H. (1996) Identification of deter-minants in the a-subunit of G, required for phospholipase Cactivation. J. Blot. Chew. 271, 5066—5072.

Voyno-Yasenetskaya T., Conklin B. R., Gilbert R. L., Hooley R..Boume H. R., and Barber D. L. (1994) G,,1, stimulates Na—Hexchange. J. Blot. Chem. 269, 4721—4724.

Wall M. A., Coleman D. E., Lee E., Ifiiguez-Lluhi J. A., PosnerB. A., Gilman A. G., and Sprang S. R. (1995) The structure ofthe G protein heterotnimer G,,113,,,. Cell 83, 1047—1058.

Wilson P. T. and Boume H. R. (1995) Fatty acylation ofa,. Effectsof palmitoylation and mynistoylation on a, signaling. J. Blot.Chew. 270, 9667—9675.

Wong Y. H. (1994) G~assays in transfected cells. Methods Enzvmol.238, 81—94.

Wong Y. H., Federman A., Pace A. M., Zachary I.. Evans T., Pouys-ségur J., and Boume H. R. (1991) Mutant a subunits of G,7inhibit cyclic AMP accumulation. Nature 351, 63—65.

Wong Y. H., Conklin B. R., and Boume H. R. (1992) G,-mediatedinhibition of cAMP accumulation. Science 255, 339—342.

Yung L. Y., Tsim S-T., and Wong Y. H. (1995) Stimulation ofcAMP accumulation by the cloned Xenopus melatonin receptorthrough G~and G, proteins. FEBS Leu. 372, 99—102.

Zigman J. M., Westermark G. T., LaMendola J., and Steiner D. F.(1994) Expression of cone tmansducin, G,a, and other G-proteina-subunit messenger nibonucleic acids in pancreatic islets. En-docrinology 135, 31—37.

J. Neurochem., Vol. 68, No. 6, /997